This report describes the certification of a reference material (IRMM-355) of Enterococcus faecalis. Certified Reference Materials (CRMs) for microbiological analysis are indispensible tools for development and validation of detection methods and for the implementation and support of internal and external quality control in the area of microbiological analysis. The content of each vial of IRMM-355 is one material sphere containing Enterococcus faecalis. The homogeneity and stability (at -20 °C and -70 °C) of the batch was assessed by counting colony forming units (cfu) per material sphere on horse blood agar (BA) and Slanetz and Bartley agar (SB agar). The material is stable when stored at a maximum temperature of -20 °C for up to 18 months. The batch was characterised by six laboratories to establish a certified value of cfu per material sphere on BA and SB agar. The certified value for BA is 890 cfu with an expanded uncertainty of 135. The certified value for SB agar is 823 cfu with an expanded uncertainty of 126. In both cases, a coverage factor k = 2 is used, corresponding to a level of confidence of about 95 %. The identity of the bacteria was confirmed by DNA sequence analysis of the coding region for the Enterococcus faecalis endocarditis antigen (efaA).